NZW x BXSB F1 (W/B F1) male mice develop systemic lupus-like disease, and several autoantibodies, circulating immune complexes, and lupus nephritis become apparent. The abnormally high incidence of degenerative coronary vascular disease with myocardial infarction and thrombocytopenia due to the presence of both platelet-associated antibodies and circulating antiplatelet antibodies in this animal has been reported. We found that W/B F1 male mice produced autoantibodies against cardiolipin (aCL) and that the titer of aCL increases with age. aCL from W/B F1 male mice were mainly IgG and binding activity to cardiolipin was aCL-cofactor (beta-2-glycoprotein I (beta-2-GPI)) dependent. We developed monoclonal aCL from these animals and examined specificity of the autoantibodies. All the mAb used reacted with the negatively charged phospholipids, cardiolipin, phosphatidylserine, and phosphatidylinositol, and some reacted with platelets and DNA. The addition of human or mouse beta-2-GPI enhanced the titer for monoclonal aCL from the W/B F1 mice. From the results of competitive inhibition enzyme immunoassay with monoclonal aCL and purified beta-2-GPI, aCL from the W/B F1 mice recognized the complex of CL and beta-2-GPI. The W/B F1 male mouse may be an appropriate model for use in studies on the pathologic significance of aCL in patients with antiphospholipid syndrome.